Systems and methods to improve organ or tissue function and organ or tissue transplant longevity

ABSTRACT

The present invention provides for systems and methods for inhaled CO therapy to prevent, attenuate, or delay processes that accelerate the loss of organ or tissue function, thereby increasing the lifespan of transplanted organs or tissues, or slowing the decline of native organs or tissues, or delaying the need for replacement of diseased native organs with organ transplants. Such biological processes that are prevented, attenuated, or delayed include chronic persistent inflammation, fibrosis, scarring, as well as immunologic or autoimmune attack.

PRIORITY

This application claims priority to U.S. Provisional Application No.61/979,712 filed Apr. 15, 2014, and U.S. Provisional Application No.61/993,140 filed May 14, 2014, the entire contents of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

In the various aspects, the invention provides systems and methods forimproving organ and tissue function, and for preventing, attenuating, ordelaying loss of organ or tissue function, including for transplantedand native organs/tissues. The systems and methods of the invention arebased on the delivery of therapeutic regimens of inhaled carbon monoxide(CO).

BACKGROUND

Chronic failure of organs and tissues often involves replacement ofhighly differentiated cells with scarring connective tissue. Forexample, chronic renal disease is characterized by progressive declineof renal function with continuous accumulation of extracellular matrix,resulting in diffuse fibrosis. Chronic failure of transplanted organsmay likewise be associated with progressive scarring, in addition toimmunological rejection that further impair the long term survival ofthe graft.

Methods and systems for preventing, delaying, or attenuating loss oforgan or tissue function, including for both native and transplantedorgans and tissues, are needed. In particular, methods and systems areneeded to prevent, attenuate, or delay chronic persistent inflammation,fibrosis, scarring, and immunological attack, among other biologicalprocesses.

SUMMARY OF THE INVENTION

The present invention in various aspects and embodiments providesmethods and systems for inhaled carbon monoxide (CO) therapy to improveorgan or tissue function and longevity, including for transplantedorgans and tissues as well as declining or diseased nativeorgans/tissues.

In various aspects, administration of an inhaled CO regimen according tothe invention prevents, attenuates, or delays processes that acceleratethe loss of organ or tissue function, thereby increasing the lifespan oftransplanted organs/tissues, or slowing the decline of nativeorgans/tissues, or delaying the need for replacement of diseased nativeorgans or tissue with transplants. Such biological processes that areprevented, attenuated, or delayed include chronic persistentinflammation, fibrosis, scarring, as well as immunologic or autoimmuneattack.

In certain embodiments, the invention provides inhaled CO therapy toreduce the incidence or reduce the likelihood of organ transplantationfailure, including preventing, attenuating, or treating acute rejection,chronic allograft rejection, vascular rejection, graft versus hostdisease, and/or delayed graft function (DGF) after kidneytransplantation. In various embodiments, the invention provides forincreased transplantation success with types of organ donations thathave been statistically less successful in terms of long term graftsurvival or avoiding DGF, thereby increasing the availability ofacceptable donor organs. In various embodiments, the invention prevents,attenuates, or delays immunologic rejection of transplanted organs ortissues, fibrosis or fibrotic scarring of transplanted organs ortissues, compromised blood supply to transplanted organs/tissues, orpersistent or chronic inflammation leading to the destruction andimpairment of transplanted organs or tissues. In some embodiments, theinvention allows reduction or elimination of immunosuppressive therapy,for example, following allograft transplantation.

In the various embodiments, CO is administered one or more ofpre-operatively, intra-operatively, or post-operatively to an organ ortissue transplant patient. CO therapy may be provided during theperi-operative period, as well substantially before or substantiallyafter the peri-operative period.

In certain embodiments, the invention provides methods for preventing,delaying, or treating fibrotic conditions of organs or tissues such asthe kidney, lung, heart, liver, pancreas, gastrointestinal tract, andskin. Exemplary conditions include chronic kidney fibrosing conditions,chronic hepatic fibrosing conditions (e.g., non-alcoholicsteatohepatitis, or NASH), chronic lung fibrosing conditions (e.g.,idiopathic pulmonary fibrosis or IPF), myocardial fibrosis, esophagealfibrosis, vascular fibrosis, and systemic diseases marked bydysregulated injury repair and/or systemic fibrosis, includingprogressive systemic sclerosis (PSS) or scleroderma.

In still other aspects, the invention provides systems and methods forsafely delivering therapeutic levels of inhaled CO to a patient, withsuch systems and methods being suitable for acute and chronic (e.g.,prolonged) treatment. For example, the invention provides systems andmethods for accurately delivering inhaled CO within its therapeuticwindow, thereby rendering inhaled CO suitable for managing chronicconditions over a prolonged period of time.

In some embodiments, the invention provides for methods and systems fordelivering inhaled CO to a patient at a constant alveolar concentrationthat maintains a selected or desired CO level in the patient for adesired period of time. These systems and methods provide fortherapeutic advantages in CO delivery for a variety of chronicconditions. In some embodiments, the CO dose is controlled by measuringinhaled CO uptake by the patient at a select CO dose for a select periodof time. The measurement of CO uptake allows for close approximation ofdiffusivity of the patient's lungs for CO (DL_(CO)), which in turnallows personalized correlation between CO dose, exposure time, andblood CO level. Thus, CO level in a patient can be carefully controlledto rapidly achieve the desired CO target, and then maintain that targetfor a desired duration. Various measurements can be used as a proxy forCO level, including carboxy-hemoglobin concentration in blood, amongothers.

For example, systems of the invention can comprise: a source of gascomprising carbon monoxide, a gas metering device operably connected tothe CO source, a gas mixing device for preparing a gas having a desiredCO concentration for delivery, a CO gas delivery unit, and a computersystem programmed to perform calculations based on relationships definedby the Coburn-Foster-Kane equation (CFK equation). For example, usingthe relationships defined by the CFK equation, the system calculatesDL_(CO) for the patient based on CO uptake (e.g., CO-Hb input values,which include a baseline CO-Hb level, and a level measured at a selectedtime point during CO administration). In some embodiments, the computersystem controls the dose of CO delivered to a patient, based on thedetermined DL_(CO) and a desired CO endpoint and duration to reach theendpoint, as well as duration to maintain the CO endpoint. In someembodiments, the system controls the CO concentration of the inspiredgas and duration, for at least two different doses of CO.

The frequency of administration of inhaled CO in accordance with theseaspects can be, for example, between once monthly to a plurality oftimes per day, to prevent, arrest, delay, or attenuate the progressionof chronic processes that lead to impaired function of transplanted ornative organs or tissues.

Other aspects and embodiments of the invention will be apparent from thefollowing detailed description.

DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary curves for percent carboxyhemoglobin saturationas a function of CO exposure duration at various CO concentration levelsfrom 8.7 ppm to 1000 ppm. Other variables of the CFK equation areprovided. From, Peterson J E, et al., Predicting the carboxyhemoglobinlevels resulting from carbon monoxide exposures, J Applied Physiol Vol.39(4):633-638 (1975).

FIG. 2 illustrates a system for delivering CO in accordance withembodiments of the invention.

FIG. 3 depicts a system for delivering CO in accordance with embodimentsof the invention.

FIG. 4 illustrates the components of the system shown in FIG. 3.

DESCRIPTION OF INVENTION

The present invention in various aspects and embodiments provides carbonmonoxide therapy to improve organ or tissue function and longevity,including for transplant organs and tissues as well as diseased ordeclining native organs. For example, administration of a CO regimen asdescribed herein can prevent, attenuate, or delay processes thataccelerate the loss of organ or tissue function, thereby increasing thelifespan of transplants, as well as delaying the need for replacement ofdiseased native organs/tissue with transplants. Such biologicalprocesses that are prevented, attenuated, or delayed include thoseinvolved in immunologic rejection of transplanted organs or tissues,fibrosis or fibrotic scarring of transplanted or native organs ortissues, compromised blood supply to transplanted or native organs ortissues, or persistent or chronic inflammation leading to thedestruction and impairment of transplanted or native organs and tissues.

In some embodiments, the patient has a condition characterized byfibrosis. Fibrosis is the formation of excess fibrous connective tissuein an organ or tissue in a reparative or reactive process. In responseto injury this is referred to as scarring. Fibrosis can obliterate thearchitecture and function of an organ or tissue. Fibrosis can beassociated with a generalized inflammatory state, with increasedcirculating inflammatory mediators. For example, in some embodiments,the patient has a condition selected from a chronic kidney fibrosingcondition, a chronic hepatic fibrosing condition (e.g., non-alcoholicsteatohepatitis, or NASH), a chronic lung fibrosing condition (e.g.,idiopathic pulmonary fibrosis or IPF), myocardial fibrosis, pancreaticfibrosis, pancreatitis, gastrointestinal fibrosis, vascular fibrosis orstrictures, or other systemic disease marked by dysregulated injuryrepair and/or systemic fibrosis, including progressive systemicsclerosis (PSS) or scleroderma. Additional conditions include cirrhosis,atrial fibrosis, esophageal fibrosis, esophageal or gastrointestinalstrictures, Crohn's Disease, Inflammatory Bowel Disease, toxicmegacolon, paralytic ileus, arthrofibrosis, arthritis, and nephrogenicsystemic fibrosis. Target organs and tissues include kidney, lung,heart, liver, gastrointestinal tract, pancreas, bone marrow, and skin.

In some embodiments, the patient has an acute or chronic inflammatory,hyperproliferative, or fibrotic condition of the lungs, as disclosed inPCT/US2014/065822, which is hereby incorporated by reference in itsentirety. Such conditions include pulmonary fibrosis (includingIdiopathic Pulmonary Fibrosis, or IPF), asthma, emphysema, ChronicObstructive Pulmonary Disease (COPD), pulmonary arterial hypertension(PAH), cystic fibrosis (CF), Acute Respiratory Distress Syndrome (ARDS),bronchiectasis, Ventilator-Assisted Pneumonia (VA), and lungtransplantation.

In still other embodiments, the patient has a condition characterized byparalysis of the gut (e.g., paralysis of peristaltic movements of thebowel). In these embodiments, without wishing to be bound by theory, COcan act as a neurotransmitter to stimulate peristaltic contractions totreat or ameliorate the condition. In some embodiments, the patient mayhave paralytic ileus (e.g., following abdominal trauma or surgery),Hirschprung's Disease, or toxic megacolon.

In some embodiments, the patient is diagnosed or considered at risk fornecrotizing enterocolitis, which is a condition typically seen inpremature infants. The timing of its onset is generally inverselyproportional to the gestational age of the baby at birth. Initialsymptoms include feeding intolerance, increased gastric residuals,abdominal distension and bloody stools.

In certain aspects, the invention provides carbon monoxide therapeuticregimens to reduce the incidence of organ transplantation failure,including preventing, ameliorating, or treating acute rejection,vascular rejection, chronic allograft rejection, and graft versus hostdisease. Historically, acute rejection may begin as early as one weekafter transplant, the risk being highest in the first three months,though it can occur months to years later. If an episode of acuterejection is recognized and promptly treated, organ failure can beprevented. Recurrent episodes can lead to chronic rejection. It isbelieved that the process of acute rejection is mediated by the cellmediated pathway. The term chronic rejection or chronic allograftrejection refers to a long-term loss of function in transplanted organs,for example, via fibrosis of the transplanted tissue's blood vessels.Graft-versus-host disease (GVHD) is a common complication following anallogeneic tissue transplant. It is commonly associated with stem cellor bone marrow transplant but the term also applies to other forms oftissue graft. Specifically, in GVHD immune cells in the tissue graftrecognize the recipient as “foreign.” The transplanted immune cells thenattack the host's body cells. In various embodiments, the recipient is arecipient of organ or tissue selected from kidney, liver, lung,pancreas, heart, bone marrow, intestinal tissue, and skin.

By receiving CO therapy, either in the first one to three months aftertransplant surgery or even with continued treatment (as described indetail herein), acute rejection of the organ or tissue can be avoided,or ameliorated, optionally together with immunosuppressive therapy. Inthese or other embodiments, the likelihood or progression toward chronicrejection is also reduced.

In some embodiments, the patient is a kidney transplant recipient. Insome embodiments, the regimen prevents, ameliorates, or treats delayedgraft function after kidney transplantation. In various embodiments, theinvention provides for increased transplantation success with types oforgan donations that have been statistically less successful in avoidingDGF or long term graft survival, thereby increasing the availability ofacceptable donor organs.

In particular, DGF after kidney transplantation may be correlated to theuse of expanded criteria donors (ECD) and donation after cardiac death(DCD). See, Siedlecki et al., Delayed Graft Function in the KidneyTransplant Am J Transplant 11(11):2279-2296 (2011). In some embodiments,the donor organ (e.g., kidney) is from a deceased donor. In someembodiments, the organ donation was after cardiac death, which has shownhigh incidence of DGF. In still other embodiments, the donation (e.g.,kidney donation) was after brain death, which also shows a highincidence of DGF, in part due to the donor's inflammatory state. In someembodiments, the donor was brain dead for at least about 10 hours, atleast about 15 hours, or at least about 24 hours before organprocurement.

In some embodiments, the CO regimen described herein allows for the useof expanded criteria donors (ECD), with lower incidence of DGF. ExpandedCriteria Donor is defined as a donor over the age of 60, or a donor overthe age of 50 with at least two of: a history of high blood pressure,creatinine of >1.5, or death from stroke. In some embodiments, the donoris over 60 years of age, over 70 years of age, or over 75 years of age.

In accordance with the invention, various organ preservation methods maybe employed, and these may include the use of CO ex vivo to enhancepreservation. For example, the inclusion of CO (e.g., 50-2000 ppm) inthe storage media in which organs to be transplanted are stored maysubstantially reduce the likelihood of oxidative damage to the organsduring storage and substantially enhances the storage time that organsto be transplanted may be safely stored without suffering irreversibleoxidative damage. Thus, in this aspect, an effective amount of CO isbubbled into storage media (with or without O₂, NO, N₂, and/or othergases) either before or preferably when an organ is first placed in themedia or shortly thereafter.

In some embodiments, CO is included in the preservation/storage media atfrom about 50 to 2000 ppm, or 50 to 1000 ppm, or 50 to 500 ppm.Generally, cold preservation is used to reduce cellular metabolic rateand thereby reduce organ damage. Adenine-containing cold preservationbuffer may be used, as well as lactobionate/raffinose solution orHistidine-Tryptophan Ketoglutarate. The donor organ may undergo machineperfusion, which may involve oxygen supplementation (with or without O₂,NO, N₂, and/or other gases). Siedlecki et al., Delayed Graft Function inthe Kidney Transplant Am J Transplant 2011. Various storage orpreservation media may be employed, with or without CO supplementation,including but not limited to Celsior solution, Perfadex, Euro-collins,and modified Euro-collins.

In various embodiments, the donor organ was preserved (with CO) for morethan about 5 hours, more than about 8 hours, more than about 10 hours,more than about 16 hours, or more than about 24 hours before thetransplant procedure.

In various embodiments, for example with respect to organtransplantation, the patient may receive CO therapy a plurality of timesduring the peri-operative period. Alternatively or in addition, thepatient may receive inhaled CO therapy substantially before and/orsubstantially after the perioperative period. In these or otherembodiments, the patient may receive CO therapy intraoperatively. Insome embodiments, CO is delivered intraoperatively, with one or more ora pre-operative or post-operative CO regimen.

For example, the recipient may be pre-conditioned with CO therapy. Therecipient may receive CO therapy from several weeks, to several days, toone or more hours prior to the organ transplant operation. In someembodiments, the recipient initiates a CO regimen, as described herein,from 1 to 8 weeks, or from 1 to 6 weeks, or from 1 to 4 weeks, or 1 or 2weeks leading up to the transplant procedure. In some embodiments, COdosing over time is beneficial in reducing baseline inflammation and/orinduction of HO-1 activity. In other embodiments, the recipient receivesinhaled CO therapy over the course of about one week leading up totransplantation. In total, prior to surgery, the patient may receivefrom 1 to about 20, or from 1 to about 15, or from 1 to about 10administrations of CO therapy (e.g., in the range of 3 to 10administrations prior to surgery). In these or other embodiments,inhaled CO is administered from 1 to 3 times on the day oftransplantation, including from about 0.5 to about 5 hours prior tosurgery. In some embodiments, CO is administered for about 1 to 3 hoursleading up to the surgery. In some embodiments, each administration ofCO is separated by at least about 6 hours, at least about 12 hours, atleast about 24 hours, at least about 36 hours, at least about 48 hours,or at least about 60 hours.

In these or other embodiments, the patient receives post-operative COtherapy. For example, in some embodiments, the recipient receives COtherapy for from 1 to 8 weeks, or from 1 to 6 weeks, or from 1 to 4weeks, or 1 or 2 weeks after the transplant procedure.

In other embodiments, the recipient receives the CO regimen over thecourse of one week, or from 1 to 3 or 1 to 4 days after transplantation.This may involve from 1 to about 20, or from 1 to about 15, or from 1 toabout 10 administrations of CO (e.g., from 3 to about 10 administrationsof inhaled CO). In these or other embodiments, inhaled CO isadministered from 1 to 3 times within the first 24 hours aftertransplantation, including being initiated at from 0.5 to 5 hourspost-surgery. In some embodiments, each administration of CO isseparated by at least about 6 hours, at least about 12 hours, at leastabout 24 hours, at least about 36 hours, at least about 48 hours, or atleast about 60 hours.

In these or other embodiments, the patient may receive intraoperative COdelivery. For example, in some embodiments, CO is administered for about1 hour leading up to the surgery, and optionally during surgery, andoptionally from one to ten times after surgery (within the first one ortwo weeks after surgery).

In some embodiments, the patient is identified as having or at risk ofhaving DGF or acute rejection post-surgery, and CO therapy isadministered post-operatively. For example, patients may be identifiedas at risk of DGF by determining the presence of slow graft function, orischemia and/or antibody induction around the time of transplantation,or other method. For example, in the case of kidney transplantation, thepatient may exhibit slow graft function, where glomerular filtrationrate and serum creatinine do not achieve normal levels but remain belowthat warranting dialysis.

In some embodiments, the patient receives inhaled CO from 2 to about 10times during the perioperative period. In some embodiments, the patientreceives inhaled CO from 2 to about 5 times during the perioperativeperiod. In some embodiments, the patient receives the first twoadministrations of inhaled CO separated by from about 36 to about 60hours, the first at the time of transplantation. In still otherembodiments, the patient receives intermittent dosing (e.g., with peaklevels of <10% carboxyhemoglobin) after the perioperative period forprophylactic treatment to prevent DGF. For example, after theperioperative period, the patient may receive inhaled CO therapy atleast once per day (e.g., 1-3 times per day), at least once per week(e.g., 1-5 times per week), or at least once per month (e.g, 1-10 timesper month). In some embodiments, the patient receives CO therapy fromabout 1 to about 5 times weekly (e.g., 1, 2, 3, 4, or 5 times per week),or receives CO therapy about every other week, or about once or twiceper month.

In the case of prophylactic or attenuating therapy for chronicconditions, either for native or transplanted organs or tissues, thepatient may receive inhaled CO therapy at least once per day, at leastonce per week, or at least once or twice per month. For example, thepatient may receive inhaled CO therapy about daily, about once or twiceweekly, or from 1-5 times monthly. In some embodiments, the patientreceives CO therapy from about once to about five times weekly (e.g., 1,2, 3, 4, or 5 times per week), or receives CO therapy about every otherweek.

In some embodiments, the patient has one or more markers of a fibroticcondition.

For example, in some embodiments, the patient has MMP1, MMP7, and/orMMP8 blood levels (e.g., peripheral blood, serum, or plasma, etc.) thatare substantially elevated compared to healthy controls. For example, insome embodiments, the baseline MMP7 levels are above about 12 ng/ml, orabove about 10 ng/ml, or above about 8 ng/ml, or above about 5 ng/ml, orabove about 3 ng/ml. In accordance with various embodiments, MMP7 levelsare tested periodically as a measure of improvement, and are maintainedat below about 8 ng/ml, and preferably below about 5 ng/ml or belowabout 3 ng/ml. For example, MMP7 levels may be substantially maintainedat about control or subclinical levels with inhaled CO therapy.Alternatively, a fibrotic condition and/or progression of a fibroticcondition can be monitored by galectin levels (e.g., one or more ofisoform 1, 2, 3, 4, 7, 8, 9, and 10) and/or soluble collagen fragmentsin the circulation (e.g., Collagen types I, II, III, and/or IV), orother marker of fibrosis. Improvement in markers can be based upon areduction from a baseline measurement taken prior to initiation of a COregimen.

During each treatment, CO gas may be administered for about 10 minutesto about 5 hours per dose, such as about 30 minutes per dose, about 45minutes per dose, about one hour per dose, or about two hours per dose.The duration of treatment in some embodiments reflects the duration thatthe target carboxy-hemoglobin level is maintained during theadministration (e.g., the steady-state level). In still otherembodiments, the duration reflects the entire administration protocol,including the time period to reach the target CO level.

There are a number of markers that can be used to monitor or measure COuptake in the patient. Although the best-known reaction of COincorporated in a human or animal body is the formation ofcarboxyhemoglobin (CO-Hb), CO can also interact with other biologicaltargets such as myoglobin (e.g., carboxymyoglobin) and cytochromeoxidase. Thus, in various embodiments one or more carboxy protein levelsare monitored, including but not limited to CO-Hb or CO-myoglobin. Insome embodiments, CO complexed with blood cells or tissue cytochromes ismeasured. Alternatively, oxygen level or pO₂ in combination with CO-Hbmay be used as a marker or end-point for CO administration. In stillother embodiments, blood pH is monitored as a marker of CO uptake. Instill other embodiments, exhaled CO or NO can be used to estimate COblood levels during CO administration.

In some embodiments, the duration and/or dose of CO is determined bycontinuous or periodic monitoring of serum oxyHb and/or CO-Hb. Forexample, CO may be administered until the patient reaches about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, or about 15% CO-Hb, with this target optionallymaintained for a period of time.

In various aspects and embodiments, the invention provides methods ofsafely delivering therapeutic levels of CO to a patient, with suchmethods being suitable for acute and chronic treatment. For example, forchronic treatment, the methods allow for safe delivery of therapeuticlevels of CO on a substantially regular basis. For example, thefrequency of administration can be between once monthly to about fourtimes weekly, to prevent, arrest, delay, or attenuate the progression ofchronic processes that lead to impaired function of transplanted ornative organs or tissues.

In various embodiments, CO-Hb is used as a marker to guide the COadministration regimen and/or the CO dosing protocol. For example, CO-Hbmay be tested before, during, or after CO administration, using a bloodtest, percutaneous device, or other device such as a pulse oximeter.CO-Hb in various embodiments can be used as a marker for the end-pointof a CO dose, and/or used to establish a CO-dosing protocol for apatient. In various embodiments, during CO administration CO-Hb ismaintained below about 20%, below about 15%, below about 12%, belowabout 10%, or below about 8%. In some embodiments, each COadministration targets a CO-Hb endpoint, which may be below about 15%,below about 12%, below about 10%, or below about 8%, or may be about 7%,about 8%, about 9%, about 10%, about 11%, or about 12%. For example, theCO-Hb endpoint may be between about 8% and about 12% (e.g., about 10%).In some embodiments, CO-Hb is maintained at the target level for aperiod of time during administration by adjusting the CO dose to achievea steady state CO level. The steady state level may be maintained forabout 30 minutes, about 45 minutes, about 1 hour, about 2 hours, orabout 3 hours. In some embodiments, for example in the case ofhospitalized patients or more advanced stages of disease, CO-Hb ismaintained at a level of from about 5% to about 15% persistently withintermittent administration of CO. In some embodiments, CO-Hb ismaintained at about 8-12% in some embodiments. For example, thefrequency of administration may be set to maintain a base CO-Hb levelover time. This level may be substantially maintained for at least aboutone week, at least about two weeks, or at least about one month.

In some embodiments, the CO administration protocol comprises at leasttwo concentration levels of CO gas; a relatively high level of CO toquickly reach a target CO blood level (e.g., as measured by CO-Hblevel), and a maintenance level of CO to maintain the CO or CO-Hbendpoint for a period of time to provide the desired therapeutic effect.In such embodiments, the administration is safe and controlled to avoidtoxic and/or undesired CO exposure, while reducing the time of theadministration procedure considerably. Further, for the treatment ofcertain chronic conditions, it may be necessary to maintain a certainlevel of CO in the blood for a period of time to achieve the desiredtherapeutic effect.

In various embodiments, the administration process comprises deliveringCO gas at a constant alveolar concentration to a patient via inhalation.The delivery of CO gas to the patient reaches a steady-state duringtreatment, where equilibrium between the alveolar concentration and thepatient's CO-Hb level is achieved. The steady-state uptake enablescontrol of the delivered CO dose, and allows for safe administration ofCO gas. In some embodiments, the steady-state mode (e.g., formaintaining a target CO-Hb level within about 6% to about 12%) iscontinued for from about 15 minutes to about 3 hours, or from about 30minutes to about 2 hours, or from about 30 minutes to about 1 hour(e.g., about 30 minutes, about 45 minutes, about 1 hour, about 1.5hours, or about 2 hours).

There are many factors that can affect the uptake of carbon monoxide bya patient via inhalation. For example, some factors are related tocharacteristics associated with the patient, including but not limitedto: changes in alveolar-capillary membrane (i.e. membrane factor); thepulmonary capillary blood volume; hemoglobin concentration; and totalblood volume. Other factors associated with the patient can include COback-pressure from endogenous CO production, and prior patient exposureto CO. The influence of these patient-related factors can vary based onthe relative health of the patient. There are also non-patient factorsthat can affect the rate and extent of uptake by the patient, namelyfactors that can be controlled or at least influenced by the nature ofthe CO delivery system. For example, the most important of these factorsis the alveolar concentration of CO. The alveolar concentration is theconcentration of CO present in the gas in a patient's lungs duringtreatment. The alveolar CO concentration is a function of the movementof gases in the lung and also the partial pressure of CO in the gases inthe lung. While the patient-related factors of CO uptake can bedifficult to measure, the alveolar concentration of CO can be heldrelatively constant through the use of the system and methods describedherein. Therefore, by controlling the alveolar concentration of CO,fluctuations in the rate of CO uptake can be minimized or avoided.

The uptake of CO in humans is mostly dependent upon the concentration ofthe inhaled gas and the diffusing capacity of the lungs. The formationof HbCO on the basis of CO exposure has been described in aphysiologically-based single-order pharmacokinetics model, and isreferred to in the literature as the Coburn-Foster-Kane equation (i.e.the CFK equation or CFKE).

CFK Equation:

$\frac{{{A\left\lbrack {{Hb}{CO}} \right\rbrack}t} - {BVco} - {PIco}}{{{A\left\lbrack {{Hb}{CO}} \right\rbrack}0} - {BVco} - {PIco}} = {\exp \left( {- \frac{tA}{VbB}} \right)}$

Where:

A=PC_(O2)/M[HBO₂]

B=1/DL_(CO)+P_(L)/V_(A)

M=ratio of the affinity of blood for CO to that for O₂

[HbO₂]=ml of O₂ per ml of blood

[HbCO]_(t)=ml of CO per ml of blood at time t

[HbCO]₀=ml of CO per ml of blood at the beginning of the exposureinterval

PC_(O2)=average partial pressure of oxygen in the lung capillaries inmmHg

Vco=rate of endogenous CO production in ml/min

DL_(CO)=diffusivity of the lung for CO in ml/min x mmHg

P_(L)=barometric pressure minus the vapor pressure of water at bodytemperature in mmHg

Vb=pulmonary blood volume

PI_(CO)=partial pressure in the inhaled air in mmHg

V_(A)=alveolar ventilation in ml/min

t=exposure time in min

exp=2.7182, the base of natural logarithms raised to the power of thebracketed expression

According to the CFK equation, the time to reach an equilibration pointbetween the alveolar concentration of CO and the body's stores can berelatively long, on the order of many hours in a healthy human (see, forexample, FIG. 2 of Peterson J E, et al., Predicting thecarboxyhemoglobin levels resulting from carbon monoxide exposures, JApplied Physiol Vol. 39(4):633-638 (1975), reproduced here as FIG. 1. Ina patient with diseased lungs, the time to reach a steady-statecondition, that is, where the blood Hb-CO level reaches a plateau, cantake even longer. However, if the HbCO level gets too high the patientcan experience severe adverse effects or even death. Further, theconcentration of CO in inhaled air can greatly affect the time needed toreach the desired steady-state concentration. For example, with a COalveolar concentration of 25 ppm, it can take about 20 hours to reach anequilibration point, while at 1000 ppm, the time to reach steady-statecan be shortened to between 2 and 3 hours. However, predictingequilibration points based on the CFK equation may be difficult,especially when Hb-CO measurements lie on the steeper region of a curveand all of the physiologic variables are unknown or cannot easily bemeasured.

The surface area of the lungs is a huge surface area, and has thegreatest influence over how much and how fast CO is absorbed by theblood. If the alveolar surface (i.e., membrane) gets thick, it slows COdiffusion. If it gets destroyed and there is less surface area, totaluptake is slowed. Of the variables from the CFK equations, only DL_(CO),starting HbCO, inspired CO, and time are important because the rest ofthem at steady-state conditions do not change much or have much impact.For example, it is sufficient to plug in reasonable estimates for thepatient (e.g., average values for the patient's age, sex, and/or weight)and leave them as constants.

In some embodiments, the CO dose is controlled by measuring inhaled COuptake by the patient at a select CO dose for a select period of time.The measurement of CO uptake allows for close approximation ofdiffusivity of the patient's lungs for CO (DL_(CO)), which in turnallows personalized correlation between CO dose, exposure time, and COlevels in the blood (e.g., CO-Hb level). According to these embodiments,the less significant physiologic variables can be bundled into a singleunmeasured factor using constants, and the actual CO uptake calculatedto allow close approximation of the only really important physiologicvariable, which is DL_(CO). Thus, CO-Hb level can be sufficientlycontrolled to rapidly achieve the desired CO-Hb target, and thenmaintain that target for a desired duration.

In various embodiments, relatively high CO concentrations (e.g., atleast 500 ppm, or at least 600 ppm, or at least 800 ppm, or at least1000 ppm, or at least 1500 ppm, or at least 2000 ppm, or at least 2500ppm) are administered to the lungs in the initial period of theprocedure in order to quickly achieve the desired CO-Hb level in thepatient. Because all of the unknown physiologic influencers of theformation of CO-Hb are unknown values during this portion of theprocedure, CO-Hb levels in the patient may be continuously orintermittently monitored to ensure that the patient's CO-Hb level doesnot exceed a safe level. In some embodiments, CO-Hb is measured exactlytwice, a baseline CO-Hb and a second measurement within about 10 minutesto about 30 minutes of CO administration. Since there may be a fine linebetween safe and harmful levels of carboxyhemoglobin, it can beimportant in some embodiments to appropriately time CO-Hb testing, so asto accurately predict Hb-CO endpoints to avoid CO toxicity.

Thus, in some embodiments, baseline CO-Hb is measured, and then theCO-Hb is measured at a known time point during CO exposure, and then byinserting the inspired CO into the equation, the equation is rearrangedand solved for DL_(CO). This DL_(CO) approximation (which is onlyslightly influenced by the unmeasured and bundled physiologic constantparameters) is plugged back into the rearranged equation and solved forthe final CO-Hb at any time point going forward using the new startingCO-Hb. The computer system (e.g., described further below) is programmedto perform these calculations, based on CO-Hb values input by a user orinput automatically through communication with sensor or diagnosticinstruments. Other values, such as desired CO-Hb target and duration,can likewise be input by the user. Similar relationships based on otherproxy's for CO level (e.g., other than CO-Hb) can be used.

The physiologically-based pharmacokinetics model associated with the CFKequation does not account for the existence of multiple physiologiccompartments in the body, that is, it does not account for physiologiccompartments other than the lungs. Benignus explored the arterial versusvenous response to inhaled carbon monoxide [Benignus et al., 1994, JAppl Physiol. 76(4): 1739-45]. According to Benignus, not all subjectsresponded alike, and while the majority of subjects followed the CFKequation, some subjects substantially deviated from the CFK model.Benignus determined that antecubital venous Hb-CO levels wereover-predicted and arterial Hb-CO levels were under-predicted,indicating the presence of at least one additional physiologiccompartment. Further, Bruce et al. [2003, J Appl Phys. 95(3):1235-47]modeled Hb-CO responses to inhaled CO, and identified five compartmentsthat can be considered in a model: lungs (alveolar), arterial blood,mixed venous blood, muscle tissue, and other soft tissues.

Therefore, in some embodiments, the CO administration protocol asdescribed further comprises modeling inhaled CO uptake by considering atleast one additional compartment other than the lungs, such as one ormore of: muscle tissue, other soft tissue, arterial blood, and/or venousblood. In some embodiments the protocol comprises calculating a CO doseusing the percentage of muscle mass in a subject as a variable.Conventional methods for CO administration use only body weight as afactor for dosage determination, which may result in missing otherrelevant factors. For example, by using only body weight, differences inthe level of muscle mass between men and women may not be consideredwhen specifying a CO dose amount for therapeutic treatment, even thoughdifferences in muscle mass can result in significant differences in COuptake and/or storage.

Further still, systems based on pulsed dosing may miss additionalrelevant factors. In systems based on pulsed dosing, a volume of CO thatis a fraction of the total dose per minute is set by the device operatorand injected into the breathing circuit. The volume fraction injected istypically determined by the patient respiratory rate, so that equalportions of the specified dose are delivered with each breath. The doseper breath is typically fixed, independent of the size of the tidalvolume, which is the volume of air displaced between normal inspirationand expiration. Therefore, when such a fixed CO volume is injected intoa varying inspired volume of air, the concentration of CO in the alveoliwill vary inversely to the size of the breath. Accordingly, at largertidal volumes, the alveolar concentration of CO will fall and the uptakeof CO will also fall. In general, patients do not breathe at a fixedtidal volume for every breath. There is a natural variation on abreath-by-breath basis, and, in addition, any variation in activity by apatient, e.g. at night when the patient is asleep, can change thealveolar concentration of CO in a pulsed-dose system. These variationscan lead to significant variation in tidal volumes, which can result ina significant change in Hb-CO levels in the patient during treatment.

Accordingly, in a pulse-based dosing system and method of COadministration, the alveolar concentration is not constant. The alveolarconcentration of one constituent in a mixture of gases is a function ofthe partial pressure of the constituent gas, i.e. the proportion of theconstituent gas to all of the other gases in the mixture multiplied bythe barometric pressure (i.e., less water vapor). For example, considera system with pulsed addition of CO gas in which a patient inhaled a 25mL bolus of a gas mixture containing 0.3% CO (3000 ppm) that was addedto 700 mL of breath of air. If the patient had a functional residualcapacity (FRC), i.e. the volume of air in the lungs at the end of anormal breath, of 1 liter, then when the inspired gas was mixed in thealveoli, the inspired gas would have been diluted to about 1.4 percentof the bolus concentration (25/(25+700+1000), or 43 ppm (a partialpressure of 3 mmHg at sea level). However, if the FRC was 400 mL insteadof 1000 mL, the CO concentration would rise to almost 1.8%, or 53 ppm(3.7 mmHg). This change represents a 23 percent increase in COconcentration that could result in a 2 percent increase in the bloodCO-Hb level, which could be enough to produce adverse health effects.Accordingly, the greater the delivered dose in a pulse-based system, thegreater the potential variability. In acute disease states and inparticular in patients on mechanical ventilators, even larger changes inFRC could be present, and such changes could result in even moresignificant swings in partial pressure that would affect uptake andmight compromise patient safety. Alternatively, when the inspired gas isprecisely premixed and delivered as a constant concentration,independent of respiratory rate, FRC, or tidal volume, there is littleor no fluctuation in alveolar gas concentration.

In certain aspects, the methods and systems provide a constant alveolarconcentration of carbon monoxide. In these aspects, the protocol safelydelivers a specified concentration of CO, for example in ppm levels, toeither mechanically-ventilated or spontaneously breathing patients. Theuse of constant alveolar concentration dosing assures that the patient'salveolar concentration will remain the same, and that the patient'sHb-CO level will reach a steady state during the treatment and enablesrelatively easy adjustments for better control of the delivered dose.Thus, in some embodiments, the concentration of CO is adjusted duringtreatment to maintain a constant alveolar concentration.

In related aspects, the invention further provides methods and systemsfor predicting CO-Hb level during CO administration. Considering thatinter-patient differences, for example diffusing capacity, cardiacoutput, endogenous carbon monoxide production and pulmonary capillaryblood volume, can result in significant differences in carboxyhemoglobinlevels for the same dosage level of CO, a method and system toaccurately predict the carboxyhemoglobin level at any point in time ofexposure would be of great value. In some embodiments, theadministration process (or the personalization of the CO regimen)comprises a reverse calculation of DL_(CO) (the Diffusing capacity orTransfer factor of the lung for carbon monoxide), to more accuratelypredict the desired CO dose. For example, a first concentration of CO isadministered for a period of time, such as from 5 minutes to about 30minutes, such as from about 10 minutes to about 25 minutes, or fromabout 15 minutes to about 25 minutes (or for about 10 minutes, about 15minutes, about 20 minutes, or about 30 minutes in various embodiments).At the end of that period CO-Hb is measured. From the actualmeasurement, DL_(CO) can be calculated from the CFK equation bysubstitution and solving for DL_(CO) because all of the other variablesare known, and DL_(CO) accounts for the balance of the difference frompredicted value. With the physiologically derived DL_(CO) (including themiscellaneous physiologic factors), one can accurately predict the CO-Hbusing the CFK equation at any point in time at the same inspired CO, ormake a change in inspired CO and predict the CO-Hb at any other point intime.

In some embodiments, a measured CO level (e.g., CO-Hb level) is inputinto the delivery system by a user or an associated CO-Hb measuringdevice, and the time and further CO dose to reach the desired end-pointis automatically adjusted by the delivery system.

For example, in some embodiments a first high concentration of CO isadministered to the patient for from 5 minutes to about 30 minutes (asdescribed above), and CO-Hb is measured either using an associateddevice or by drawing blood for testing. From the CO-Hb measurement, thedose of inhaled CO to reach the desired CO-Hb endpoint at a particulartime is determined. This determination can comprise calculating DL_(CO).In some embodiments, CO-Hb is measured after about 15 to about 25minutes of the initial CO dose (e.g., after about 20 minutes of theinitial CO dose), to allow saturation of physiological compartments suchas muscle, thereby providing a more accurate estimate of the CO doseneeded to reach the desired CO-Hb endpoint. Once the patient ispredicted to reach the CO-Hb endpoint, the CO dose is adjusted to thedose determined to maintain a steady-state CO-Hb level. Thesepredictions can be made using relationships defined by the CFK equationas disclosed herein.

In some embodiments, each administration of CO is a predeterminedregimen to reach a selected CO-Hb endpoint (e.g., steady-stateconcentration), and maintain that end-point for a period of time. Thisregimen may be empirically tested for the patient, and determined basedon a set of criteria, and then subsequently programmed into the deliverysystem. In these embodiments, cumbersome and invasive blood tests areavoided, which further renders the treatment suitable for home care.Further, in some embodiments, the method or system does not rely oncontinuous CO or CO-Hb measurements, but relies on a specified regimenpersonalized for the patient. Thus, in some embodiments, CO dosingscheme (e.g., ppm over time, including steady-state administrationsteps) is determined in a personalized manner in a clinical setting withCO-Hb testing (e.g., blood test), and DL_(CO) calculation, and theselected dosing schedule used going forward (either in the clinic oroutpatient setting) without CO-Hb monitoring. In some embodiments, theCO-Hb is tested after administration at least once per year, or onceevery 6 months, or every other month, or once a month, to ensure thatthe dosing regimen remains appropriate for the patient, based on, forexample, improving or declining health (e.g., lung function). In someembodiments, a less invasive pulse oximeter can be used to monitor COlevels when using a personalized regimen as described herein.

In various aspects, the invention uses systems to reliably control theCO administration process. For example, the methods may employ a COdosing system to regulate the quantity of carbon monoxide which isdelivered from a carbon monoxide source to the delivering unit. Invarious embodiments, the system comprises a sensor that determines theconcentration of carbon monoxide in the blood of the patient, includingspectroscopic or other methods, and/or means to measure carbon monoxidein the gas mixture expired from a patient (e.g., by spectroscopicmethods or gas chromatography). The system may further comprise acontrol unit for comparing the actual CO blood concentration with apreset desired value, and subsequently causing the dosing unit toregulate the amount of carbon monoxide delivered to the patient toobtain a concentration in the patient's blood corresponding to thepreset desired value. The control unit may perform a program control, asensor control, or a combined program/sensor control.

CO-Hb levels can be determined by any method. Such measurements can beperformed in a non-invasive manner, e.g., by spectroscopic methods,e.g., as disclosed in U.S. Pat. Nos. 5,810,723 and 6,084,661, and thedisclosure of each is hereby incorporated by reference. Invasive methodswhich include the step of taking a blood sample, are employed in someembodiments. An oxymetric measurement can be performed in someembodiments, e.g., as disclosed in U.S. Pat. No. 5,413,100, thedisclosure of which is hereby incorporated by reference.

There is an equilibrium regarding the distribution of carbon monoxidebetween blood and the respired gas mixture. Another method fordetermining the blood concentration of CO is the measurement of thecarbon monoxide concentration in the expired air of a patient. Thismeasurement may be done by spectroscopic methods, e.g., by ultra-redabsorption spectroscopy (URAS), or by gas chromatography. This method ofdetermination is well-established in medical art for the determinationof the diffusing capacity of the lungs of a patient.

In some embodiments, the CO administration procedure comprises: settinga target Hb-CO level in the blood of the patient to be treated;administering CO gas at a first concentration while measuring the HbCOlevel in the patient's blood; reducing the CO level to a secondconcentration while continuing to monitor the patient's HbCO level; andcontinuing the administration of CO gas at the second concentration fora desired period of time, referred to herein as steady-state mode. Forexample, CO gas may be delivered via inhalation for a relatively briefinitial period, for example 30 minutes to 1 hour, at an inhaled COconcentration of 100 to 2000 ppm until a desired blood level of CO isreached, for example about 7%, about 8%, about 9%, or about 10% HbCO, orother target concentration described herein. The time to reach thetargeted value can vary significantly according to the patient's lungfunction or other factors, and methods for predicting the time requiredto reach the target blood level can be inaccurate or inconsistent. Insome embodiments, the CO gas is delivered at an initial CO concentrationuntil the desired HbCO level is achieved, instead of setting a specifictime period for the CO delivery at the first CO concentration.

In one embodiment, the concentration of the CO being administered can beadjusted during administration based on real-time feedback from a pulseoximeter, or any other type of sensor that can directly or indirectlymeasure CO levels in a patient's blood. In such an embodiment, a targetlevel of HbCO is set instead of setting a target level for the COconcentration being administered. The CO concentration can beautomatically adjusted by the control system, depending on how thepatient's HbCO level are responding to the CO concentration beingdelivered. For example, if the patient's HbCO level is increasing fasterthan expected, in comparison to pre-set reference parameters, thecontrol system can lower the CO concentration being administered. In oneembodiment, the control system uses the CFK equation to calculate theDL_(CO) and then calculates the change in inspired CO concentrations.

In one embodiment, once the desired HbCO level in the patient's blood isachieved, CO gas is delivered to the patient at a second, lowerconcentration for a desired period of treatment time (e.g., from about30 minutes to about 3 hours). This period of delivery at the second COconcentration is generally referred to herein as the steady-statedelivery mode. In such embodiments, the CO concentration is reduced tothe level needed to maintain the target CO-Hb level at steady-statewithout exposing the patient to toxic levels of CO.

An exemplary system for administering CO (100) is illustrated in FIG.2-4. CO gas source (110) provides a gas mixture comprising CO to ametering device (120) through connection (115). Gas is mixed fordelivery with air from air source (130), through connections (125) and(135) by a delivery gas mixing device (140). Mixed gas is delivered at aconstant dose to the patient (150) through the delivery unit (145). Gasdelivered to the patient is measured, for example, by a gas samplingport (127.) The system may further comprise a sensor measuring the flowof inspired room air, such as flow sensor (137). The system may furthercomprise a computer (e.g., Central Processing Unit and software)calculating DL_(CO) based on CO-Hb values provided by user input or byconnection to a CO-Hb sensor, and using other measured or input valuesfor the CFK equation. The computer system may further provide foradjustment of the CO dose, by calculating (based on the DL_(CO)) the COdose needed to reach a determined CO-Hb end-point in a specifiedduration. The computer system may further provide for determining asteady-state CO dose (e.g., that maintains a CO-Hb target). In someembodiments, the computer system controls a CO dosing regimen for aparticular patient, by delivering a preset “high” amount of CO for aperiod of time, and then delivering a second lower dose of CO for a setduration. The high dose is determined in a clinical setting as the doseneeded for the patient to achieve the desired CO-Hb end-point, and thesecond dose is determined clinically as a steady-state dose for thepatient at the desired CO-Hb level. The required values in terms of COconcentration and duration of each dose can be input by the user (e.g.,clinician).

In one embodiment, the system also comprises a pulse oximeter sensorthat measures the HbCO level in a patient's blood. By non-limitingexample, the pulse oximeter may be a Massimo RAD57 pulse oximeter. Inanother embodiment, the system comprises a sensor that measures theconcentration of CO gas in the patient breathing circuit. In yet anotherembodiment, the system of the present invention comprises any type ofsensor, other than a pulse oximeter, that is suitable for measuring ordetermining the HbCO level in a subject's blood. By non-limitingexample, the sensor may be an Instrumentation Laboratories IL-182CO-Oximeter.

In one embodiment, the system or method involves at least one centralprocessing unit (CPU) or microprocessor for use in monitoring orcontrolling the CO gas concentration in the breathing circuit, the HbCOlevel in the patient, or any other variable necessary for operation ofthe system and methods described herein. In cases where it is desired tomaintain a target HbCO level in a patient after the target level isreached, the device can automatically decrease the inspired CO gasconcentration to the level required to maintain the desired steady-stateHbCO concentration. The system may also comprise alarm or warningsystems that can trigger warning messages or an automated shut-off, asdescribed herein. In one embodiment, the measured HbCO values arecontinuously read by a CPU, and if the HbCO level rises above thepre-set threshold, the CPU can sound an alarm, display a warning messageon the control unit, and/or send a signal to turn off delivery of CO gasto the breathing circuit.

In one embodiment, the system has at least two CPUs, wherein one CPU isused for monitoring the mixing of gases, for example air and CO, and theflow of CO-containing gas to the patient. In such an embodiment, asecond CPU monitors other variables, for example the concentration of COor oxygen in the inspired gas, the HbCO level measured by the pulseoximeter, or any other variable associated with the system. Further, thesystem may monitor the pressure in one or more gas source tanks feedinggas to the control system of the present invention, in order to assurethat continuous therapy, i.e. gas flow, is provided.

In some embodiments, the system and method may further comprise otherfeatures, such as an alarm or warning system, an automatic shutofffeature, or an automated transition to a steady-state delivery mode. Inone embodiment, when the CO-Hb level in the patient or the COconcentration in the breathing circuit is greater than the desiredtarget level, the system of the present invention can institute an alarmor warning message to alert the operator, patient, or other person, ofthe deviation of the measured variable from a set point or target level.The alarm can be in the form of any visual, audio, or tactile feedbackthat would be suitable for informing a person of the deviation. Inanother embodiment, the system and method comprises an automatic shutofffeature that stops delivery of CO gas to the patient when the CO-Hblevel or CO concentration in the breathing circuit exceeds a specifiedlevel.

In yet another embodiment, the system or method of the present inventioncomprises an automated transition to a steady-state delivery mode,wherein the concentration of CO gas being delivered to the patient isautomatically reduced to a lower concentration once the desired level ofCO-Hb in the patient has been achieved.

In various embodiments, the CO gas is administered at from 50 to 500 ppmCO for the steady-state mode. For example, the CO gas may beadministered at from 50 to 300 ppm of CO, or from 50 to 150 ppm CO, orfrom 50 to 100 ppm CO in some embodiments for the steady-state mode. Insome embodiments, CO gas is administered at less than 100 ppm. In otherembodiments, the CO gas may be administered at from 100 to 400 ppm, suchas from 200 to 300 ppm or from 250 to 300 ppm. In some embodiments, theCO gas is more than 200 ppm for steady-state mode.

In some embodiments, oxygen gas is delivered simultaneously orseparately from CO to reduce CO toxicity or provide an enhanced effect.For example, oxygen gas may be delivered to the patient from 1 to 7times per week, with or between CO treatments, and for from about 10minutes to about 1 hour per treatment. When provided simultaneously, theCO:O₂ ratio may be from 100:1 to 1:100, or from 10:1 to 1:10, or from5:1 to 1:5, or from 2:1 to 1:2 in some embodiments. In some embodiments,O₂ is delivered separately, and between CO administrations. For example,O₂ may be administered prior to CO administration by about 24 hours orless, or about 12 hours or less, or about 6 hours or less, or about 2hours or less, or about 1 hour or less. In these or other embodiments,O₂ may be administered after CO administration by about 24 hours orless, or about 12 hours or less, or about 6 hours or less, or about 2hours or less, or about 1 hour or less. O₂ may be administered beforeand after one or more CO administration(s). O₂ may be administered for aduration of from about 15 minutes to about 2 hours, including from about30 minutes to about 1 hour.

In some embodiments, the CO therapy is provided to a patient thatreceives NO therapy, either simultaneously or separately. In someembodiments, without wishing to be bound by theory, NO therapy acts as avasodilator to improve or enhance delivery of CO to organs and tissues.

Gaseous CO-containing compositions may be prepared by mixingcommercially available compressed air containing CO (generally about 1%CO) with compressed air or gas containing a higher percentage of oxygen(including pure oxygen), and then mixing the gases in a ratio which willproduce a gas containing a desired amount of CO. Alternatively,compositions may be purchased pre-prepared from commercial gascompanies. In some embodiments, patients are exposed to oxygen (O₂ atvarying doses) and CO at a flow rate of about 12 liters/minute in a 3.70cubic foot glass exposure chamber. To make a gaseous compositioncontaining a pre-determined amount of CO, CO at a concentration of 1%(10,000 ppm) in compressed air is mixed with >98% O2 in a stainlesssteel mixing cylinder, concentrations delivered to the exposure chamberor tubing will be controlled. Because the flow rate is primarilydetermined by the flow rate of the O2 gas, only the CO flow is changedto generate the different concentrations delivered to the exposurechamber or tubing. A carbon monoxide analyzer (available from InterscanCorporation, Chatsworth, Calif.) is used to measure CO levelscontinuously in the chamber or tubing. Gas samples are taken by theanalyzer through a portion the top of the exposure chamber of tubing ata rate of 1 liter/minute and analyzed by electrochemical detection witha sensitivity of about 1 ppb to 600 ppm. CO levels in the chamber ortubing are recorded at hourly intervals and there are no changes inchamber CO concentration once the chamber or tubing has equilibrated. COis then delivered to the patient for a time (including chronically)sufficient to treat the condition and exert the intended pharmacologicalor biological effect.

In a preferred embodiment, the CO-containing gas is supplied in highpressure vessel containing between about 1000 ppm and about 100,000 ppm,such as about 3,000 to 8,000 ppm, such as about 4,000 to about 6,000ppm, such as about 5,000 ppm, and connected to a delivery system. Thedelivery system can measure the flow rate of the air that the patient isbreathing and can inject a proportionally constant flow rate of theCO-containing gas into the breathing gas stream of the patient so as todeliver the desired concentration of CO in the range of 0.005% to 0.05%to the patient to maintain a constant inhaled CO concentration.

In another embodiment, the flow of oxygen-containing air that isdelivered to the patient is set at a constant flow rate and the flowrate of the CO-containing gas is also supplied at a constant flow ratein proportion to the oxygen-containing air to deliver the desiredconstant inhaled CO concentration.

The pressurized gas including CO can be provided such that all gases ofthe desired final composition (e.g., CO, He, Xe, NO, CO₂, O₂, N₂) are inthe same vessel, except that NO and O₂ cannot be stored together.Optionally, the methods of the present invention can be performed usingmultiple vessels containing individual gases. For example, a singlevessel can be provided that contains carbon monoxide, with or withoutother gases, the contents of which can be optionally mixed with thecontents of other vessels, e.g., vessels containing oxygen, nitrogen,carbon dioxide, compressed air, or any other suitable gas or mixturesthereof.

Carbon monoxide compositions according to embodiments of the presentinvention can comprise 0% to about 79% by weight nitrogen, about 21% toabout 100% by weight oxygen and about 1000 to about 10,000 ppm carbonmonoxide. More preferably, the amount of nitrogen in the gaseouscomposition comprises about 79% by weight, the amount of oxygencomprises about 21% by weight and the amount of carbon monoxidecomprises about 4,000 to about 6,000 ppm.

The amount of CO is preferably at least about 0.001%, e.g., at leastabout 0.005%, 0.01%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%,0.10%, 0.15%, 0.20%, 0.22%, or 0.24% by weight. Preferred ranges of COinclude 0.005% to about 0.24%, about 0.01% to about 0.22%, about 0.015%to about 0.20%, and about 0.025% to about 0.1% by weight. It is notedthat gaseous CO compositions having concentrations of CO greater than0.3% (such as 1% or greater) may be used for short periods (e.g., one ora few breaths), depending upon the application.

A gaseous CO composition may be used to create an atmosphere thatcomprises CO gas. The gases can be released into an apparatus thatculminates in a breathing mask or breathing tube, thereby creating anatmosphere comprising CO gas in the breathing mask or breathing tube,ensuring the patient is the only person in the room exposed tosignificant levels of CO.

CO levels in an atmosphere can be measured or monitored using any methodknown in the art. Such methods include electrochemical detection, gaschromatography, radioisotope counting, infrared absorption, colorimetry,and electrochemical methods based on selective membranes (see, e.g.,Sunderman et al., Clin. Chem. 28:2026 2032, 1982; Ingi et al., Neuron16:835 842, 1996). Sub-parts per million CO levels can be detected by,e.g., gas chromatography and radioisotope counting. Further, it is knownin the art that CO levels in the sub-ppm range can be measured inbiological tissue by a midinfrared gas sensor (see, e.g., Morimoto etal., Am. J. Physiol. Heart. Circ. Physiol 280:H482 H488, 2001). COsensors and gas detection devices are widely available from manycommercial sources.

In some embodiments, the CO gas is administered to the patient byventilator. In some embodiments, the CO gas is administered to thepatient by an extracorporeal perfusion machine. In some embodiments, thepatient is able to spontaneously breathe, and the CO gas is administeredwithout any ventilation assistance.

In some embodiments, MMP7 levels are tested at least once weekly or oncemonthly, and the patient's treatment adjusted to substantially maintainMMP7 levels near subclinical levels (e.g., less than about 6 ng/ml orless than about 5 ng/ml or less than about 4 ng/ml), or to reduce othersurrogate markers of fibrosis as described herein.

In some embodiments, the patient is undergoing therapy with one or morepharmaceutical interventions, which provides additional and/orsynergistic benefits with the CO regimen.

For example, in some embodiments, the patient receives nitric oxidetreatment, in addition to CO. In some embodiments, the patient isundergoing therapy with one or more of the following: one or moreanti-inflammatory and/or immunomodulating agents, an anticoagulant,endothelin receptor antagonist, vasodilator, antifibrotic, cytokineinhibitor, and kinase inhibitor.

In various embodiments, the patient is undergoing therapy with acorticosteroid, such as prednisone or prednisolone. In some embodiments,the patient is undergoing treatment with azathioprine and/orN-acetyl-cysteine (NAC). In some embodiments, the patient is undergoingdouble or triple therapy with a corticosteroid (e.g., prednisone),azathioprine, and/or NAC. In still other embodiments, the patient isundergoing treatment with an antifibrotic, such as pirfenidone orInterferon-γ, or TNF-α inhibitor (e.g., etanercept). In these or otherembodiments, the patient is undergoing treatment with one or moreanticoagulants, such as warfarin or heparin. In these or otherembodiments, the patient is undergoing treatment with one or moretyrosine kinase inhibitors, such as BIBF 1120 or Imatinib. In these orother embodiments, the patient is undergoing treatment with one or morephosphodiesterase inhibitors, such as sildenafil, or endothelin receptorantagonist, such as bosentan, ambrisentan, or macitentan. Othertherapies that may provide synergistic or additive results with COtherapy include inhibitors of IL-13, CCL2, CTGF, TGF-β1, αvβb integrin,LOXL (e.g., neutralizing monoclonal antibody against IL-13, CCL2, CTGF,TGF-β1, αvβb integrin, LOXL).

In some embodiments, the patient is undergoing therapy with one or morepharmaceutical interventions, such as NO to assist in ischemicpreconditioning, or other agents that reduce inflammation, inducevasodilation, or induce immunosuppression. Exemplary pharmaceuticalinterventions that can be synergistic with CO (or combination of gasesdescribed herein) in various embodiments include: Perfeindone,Nintedanib, Simtuzumab, STX-100 (Biogen), FG-3019 (Fibrogen),Tralokinumab, BMS-986020 (BMS), and Lebrikizumab, among others. Invarious embodiments, the patient undergoes additional therapy that actsas a TGF-β inhibitor, antioxidant, and/or anti-inflammatory; tyrosinekinase inhibitor suppressing FGFR, PDGFR and/or VEGFR; inhibitor ofLOXL2 and/or TGF-beta signaling; inhibitor of TGF beta activation;inhibitor of Connective Tissue Growth Factor (CTGF), inhibitor of IL-13;or an antagonist of lysophosphatidic acid receptor.

Additional agents that are synergistic with CO in some embodiments,including therapy with an endothelin receptor antagonist,phosphodiesterase inhibitor, calcium channel blocker (e.g.,dihyrdopyridine), and adenosine A1 receptor antagonist (e.g.,rolofylline). Other recipient therapies that can be synergistic with COin some embodiments include anti-inflammatory agents and apoptosisinhibitions (e.g., rPSGL-Ig, CXCR inhibitor, Diannexin).

In some embodiments, the recipient undergoes immunosuppressant therapy,such as with basiliximab, thymoglobulin (anti-thymocyte globulin orATG), daclizumab, and/or alemtuzumab. In some embodiments, the patientundergoes chronic immunosuppression with a calcineurin inhibitor,sirolimus or everolimus, or Trimetazidine. In some embodiments, thepatient is able to reduce or eliminate immunosuppressive therapy with COtreatment as described herein.

EXAMPLES Example 1 Study of Back Calculation of DLCO and Predicting COHbat 60 Minutes

To test the ability to predict CO-Hb levels at 60 minutes based on bloodmeasurements of COHb at earlier time points, an experiment was performedin a S. pneumoniae model induced in four juvenile baboons. Usingmeasured COHb levels after 10, 20, 30, 40, and 50 minutes of 200 ppm COadministration, a computer program generated in MATLAB (Mathworks) wasused to back calculate the estimated DL_(CO) (including unmeasuredphysiologic variables) using the CFK equation (Coburn et al. JCI, 43:1098-1103, 1964; Peterson et al. JAP, Vol. 39, No. 4, 633-638, 1975).Then using the estimated DL_(CO) and measured time point CO-Hb levels,the computer then used the CFK equation to predict the CO-Hb level aftera 60 min. CO exposure. There was good correlation between the predictedand measured COHb levels (Table below). It was determined that thismethod can be used to predict the 60 min CO-Hb level with high accuracy(R2=0.9878) using the 20 min COHb level.

Time Point 60 Min predicted COHb (Min) minus actual COHb SD 95% CI 10−0.777% 1.07 (−3.4, 1.8) 20 0.28% 0.43  (−0.4, 0.97) 30 −0.05% 0.18(−0.33, 0.23) 40 −0.13% 0.06  (−0.23, −0.03) 50 −0.11% 0.05  (−0.2,−0.02)

Example 2 A Randomized, Single-Blind Placebo-Controlled Study toEvaluate the Safety and Efficacy of Inhaled Carbon Monoxide in thePrevention of Delayed Graft Function (DGF) in Allogeneic RenalTransplant Recipients Objectives:

A primary objective is to evaluate the safety and tolerability of CarbonMonoxide administered by inhalation in a concentration of 250 ppmintra-operatively at the time of renal graft reperfusion and in theimmediate days post-transplant. CO will be administered forapproximately 1 hour to achieve a target blood carboxyhemoglobin level(COHgb) of 8-10%. Safety and tolerability at the time of transplant willbe assessed by the occurrence of Adverse Events (AEs) and by themonitoring of vital signs, blood oxygenation, serum hematology andchemistry, cardiac status by both telemetry (intraoperatively) andelectrocardiograms (ECGs), and clinical evaluation of neurocognitivestatus by a battery of standard evaluation tools that assessconcentration, reaction time, and memory.

Another primary objective is to evaluate the efficacy of CO bydetermining its impact on delayed graft function (DGF), measured in theweek following transplantation.

Other objectives include: (1) Characterizing the pharmacokinetics (PK)of COHgb in renal transplant subjects following inhalation of CO; (2)evaluating the efficacy of inhaled CO by measuring creatinine clearanceand the trajectory of estimated glomerular filtration rate (eGFR)improvement in the week following transplantation; (3) evaluating theassociation between safety and tolerability outcomes to inhaled CO dosesand COHgb levels (as assessed by analysis of venous blood samples).

An additional objective is to assess the change in renal functionpost-transplant by serum blood urea nitrogen (BUN) and serum creatinineAssess markers of renal ischemic reperfusion injury and DGF by measuringboth urine and serum biomarkers in the week observation periodpost-transplant. These could include, (but not be limited to) urineIL-18 and NGAL (neutrophils gelatinase-associated lipocalin) levels aspotential markers for the incidence of DGF. Other potential markers toevaluate or identify recipients at risk for DGF include mRNA expressionprofiles, miRNA serum or tissue profiles, the presence of certain SNPs,and/or the MRP2 gene or its expression.

Methods:

Patients scheduled to receive allogeneic transplants 18-70 years of ageat participating centers will be the targeted population for this study.This study will be conducted as a single-blind, placebo-controlled,study. Participating subjects will be randomized to receive CO orplacebo (room air) intraoperatively, with inhaled CO therapy to startintraoperatively at or just before the time of renal graft reperfusion.CO will be administered as 250ppm CO blended into the patient'sbreathing circuit from a gas source containing 5000 ppm CO, 21% O2,balance Nitrogen mixture. Subjects will be dosed intraoperatively,supervised by the operative anesthesiologist, and will be administratedas the inhaled CO gas for approximately 1 hour to achieve a targetcarboxyhemoglobin level of 8-10%. Intraoperative dosing will be donewhile the patient is intubated.

On the second post-operative day, between 36 and 60 hours after kidneygraft reperfusion, patients will be given a second inhaled CO or placebodose using the same delivery system via mask or mouthpiece whilebreathing spontaneously. This dose will again be with 250 ppm of CO, 21%O2, balance nitrogen, for one hour, again targeting a blood COHgb levelof 8-10%. A total of 200 subjects receiving cadaveric kidney transplantsand fulfill the entry criteria will be allocated to receive CO orplacebo. Follow-up visits will be planned for Day 7 (or assessmentconducted in hospital, if the subjects remains in the hospital 7 daysafter transplant), Day 14, and 3 months after the administration ofstudy drug.

A total of 200 subjects, allocated equally between the CO and placebotreatment arms, will be studied. Assessment of DGF, the primary efficacyoutcome will occur at 7 days. Subjects will return for safetyassessments for study purposes at 14 days, and again 3 monthspost-transplant. Efficacy assessments of renal function will occur atmultiple times during the 7 days after transplant, and at the 14 day and3 month follow-up visit. These will include serum creatinine and BUNmeasurements, as well as creatinine clearance to estimate GFR.

Number of Subjects (Planned):

200 subjects receiving allogeneic cadaveric renal transplants willcomprise the study population. One hundred subjects each will berandomly allocated to inhaled CO and placebo treatment.

Inclusion Criteria:

Subjects 18-70 years of age receiving cadaveric kidney transplants;

Subjects who were dialysis-dependent prior to transplant;

Subjects undergoing first renal transplant

Exclusion Criteria:

Subjects who had prior kidney transplant;

Subjects with history of malignancy within the prior 5 years, other thanbasal cell skin cancers;

Subjects with significant pre-transplant anemia, whose serum Hgb isconsistently <10.0 g/dl;

Subjects who, in the opinion of the investigator, have unstable medicalissues rendering them at significantly greater risk for adverse eventsin the peri-operative period;

Patients with significant pulmonary gas exchange compromise orconditions, such as interstitial lung disease or severe emphysema, andall subjects who consistently have baseline oxygen saturations on roomair of <90%;

Subjects with baseline carboxyhemoglobin (CO Hgb) levels >2%;

Subjects whose body-mass index (BMI) exceeds 35;

Subjects receiving a kidney graft from a donor younger than 35 years oldAND with a cold ischemic time less than 16 hours. This purpose of thiscriterion is to exclude subjects undergoing transplant who have very lowlikelihood of developing delayed graft function;

Subjects who are currently or in the past 60 days have been onexperimental or as yet unapproved medications, or who have been activelyenrolled in a clinical trial exploring new therapies;

Patients with a calculated panel reactive antibodies (PRA), calculatedby single bead antigen of >=10%;

Graft kidneys that come from donors who are: (1) less than 30 years old,(2) who are from donors less than 40 years old with cold ischemia times<12 hours; (3) Donors whose pre-donation serum creatinine >3.0 mg/dl

Pre-Transplant Assessments:

These assessments of donor, graft, and recipient (subject) factors willbe included in trial database to allow stratified analyses by riskstrata and/or as predictor variables in multi variable models to controlfor their impact on the occurrence of DGF.

Donor Factors:

Age

Cause of Death

Pre-morbid conditions

Hypertension

Diabetes

Weight

Terminal Creatinine

Donation after Cardiac Death

Donation after Brain Death

Expanded vs. Standard Donor Criteria

Use of anticoagulation therapies

Graft Factors:

Warm Ischemia Time

Cold Ischemia Time

Machine Perfused Post vs. Cold Storage Post Harvest

Pump parameters if machine perfused

Preservation Solution & Buffer: UW, HTK,

Time 0 biopsy

Recipient Factors:

Age

Gender

Race

Pre-Morbid Conditions

Diabetes

Hypertension

CHF

Panel Reactive Antibodies

Alloantibodies and Crossmatch Results

BMI

Duration of Dialysis Prior to Transplantation

Investigational Product, Dosage:

For this Phase 2 trials, a standardized gas blend will be used whichconsists of 5000 parts-per-million (ppm) Carbon Monoxide in an Airmatrix. The Air component is comprised of 21% Oxygen and a Nitrogenbalance. The Oxygen and Nitrogen comply with the monograph requirementslisted in the United States Pharmacopeia (USP) and National Formulary(NF), respectively, both of which are recognized as legal standards bythe FDA.

The Carbon Monoxide ingredient is Grade 3 (99.9%), the standard gradefor medical gas applications such as lung diffusion mixtures. It isblended gravimetrically with the Air matrix to a tolerance of ±5%relative (or 4750-5250 ppm). For the Phase 2 trials, the nominal mixture5000 ppm CO will allow for dilution through the delivery device at lowerconcentrations (250 ppm).

Duration of Treatment:

Inhalation for approximately 1 hour (250 ppm inhaled CO or placebo)administered intraoperatively via the ventilator circuit at the onset orprior to the onset of renal graft revascularization, until the subjectattains a CO-Hb level in the 8-10% target range. Similarly, on day 2post op, subject will breath 250 ppm inhaled CO vs. placebo, for 1 hourvia face mask, again targeting attained CO Hgb level of 8-10%.

Oxygen (O₂) 21% with balance nitrogen (N₂) will be used as placebo andadministered as a gas for inhalation according to the same regimen asthe active study drug.

Evaluation Criteria Safety:

Adverse events

Discontinuations from Study

Vital signs (temperature, blood pressure, heart rate, respiratory rate)

Laboratory Values

Hematology

Serum Chemistry

Urinalysis

Electrocardiogram (ECG)

O₂ saturation by Pulse Oxymetry

COHbg from venous blood samples

Neurocognitive testing battery

Efficacy:

Occurrence of delayed graft function (defined below) in week followingtransplant

Markers of Renal Function in the week following transplant, and during 3month follow-up

Creatinine Clearance/estimated glomerular filtration rate (GFR)

Serum Creatinine, BUN

Urine Output

Dialysis

Serum Biomarkers

Pharmacokinetics:

The concentration profiles of COHbg in blood will be evaluated for eachsubject. The PK parameters of COHb will be assessed for area under theblood concentration versus time curve (AUC0-t and AUCinf), the maximumblood concentration (Cmax), the time when the maximum bloodconcentration is achieved (Tmax), terminal half-life (t½), and apparentvolume of distribution at steady state (Vss/F).

Delayed Graft Function Definition:

The Renal Transplant Literature has employed a number of differentdefinitions of Delayed Graft Function (DGF). In this study, a modifieddialysis-based definition will be employed. The components of thisdefinition are as follows: (1) The necessity for dialysis in the 7 daysfollowing reperfusion of donor kidney, other than dialysis on a singleoccasion for the purpose of control of plasma volume or hyperkalemiarather than persistent or ongoing renal function impairment and/or (2)At 48 hours post-transplant, the failure of the estimated GFR(creatinine clearance in ml/min/m2) to improve from that observed in thefirst 6 hours immediately post-reperfusion of donor kidney.

Endpoints Primary Efficacy Endpoint:

Proportion of transplant subjects satisfying DGF definition at 7 dayspost-transplant

Secondary Efficacy Endpoints:

Trajectory of change/improvement of estimated GFR over the 7 daysfollowing reperfusion of donor kidney

Trajectory of change/improvement of serum creatinine over the 7 dayspost-reperfusion of donor kidney

Trajectory of change in estimated GFR over the first 48 hourspost-reperfusion of donor kidney

Creatinine reduction ratio over 2 days post-transplant: [Creat day1]−[Creat day 2]/[Creat day 1]

Proportion of subjects with reductions in Serum Creatinine of 10%/day ineach of the first 3 days after transplant

GFR on Day 7 post-reperfusion of donor kidney

Serum Creatinine on Day 7 post reperfusion of donor kidney

Duration of Dialysis for Subjects who require it

Estimated GFR at the 3 month follow-up

Serum Creatinine at the 3 month follow-up

Proportion of Subjects with Graft Loss/Graft Survival at the 3 monthfollow up

Proportion of subjects with Primary Non-Function (PNF) of Graft

Safety:

Adverse Events

SAEs (Re-hospitalization, prolongation of hospitalization, lifethreatening events

Respiratory AEs, especially in during post-transplant week in hospital

Neurocognitive AEs

Discontinuations

Safety Laboratory Abnormalities

Allowed Medications:

All medications that are typically or routinely used in the care ofpatients undergoing renal transplant will be allowed in this trial.Therapies that are routinely started in the immediate post-operativeperiod, including calcineurin inhibitors (CNIs) such as Tacrolimus andCyclosporine, will also be allowed. Their use and doses will be recordedand included in the database to permit their use as predictor orstratifying variables in the analysis of DGF.

The mTOR inhibitors sirolimus and everolimus are excluded. Subjects whoare treated with these therapies will be excluded from a ‘Per Protocol’analysis, as will patients for which therapy permitted CNI inhibitorsare initiated sooner than 48 hours after kidney graft reperfusion.

Data Analysis and Statistical Methods:

Adverse events will be coded using MedDRA (most current version) andsummarized by treatment (Inhaled CO vs. PBO) for the number of adverseevents. A by-subject treatment emergent adverse event listing includingthe verbatim term, preferred term, treatment, severity, and relationshipto treatment will be provided. Treatment-emergent adverse events will bedefined as those occurring from the time of intraoperative COadministration until 7 days post-transplant. The number of subjectsexperiencing adverse events and the number of adverse events will besummarized by treatment using frequency and counts. Concomitantmedications will be coded using the WHO Drug Dictionary (most currentversion).

Safety data including renal function parameters and clinical laboratoryevaluations, vital signs, electrocardiograms, and oxygen saturationassessments will be summarized by treatment and time point ofcollection. Descriptive statistics will be calculated for quantitativesafety data and frequency and counts will be compiled for classificationof the qualitative safety data. In addition, a mean change from baselinetable will be provided for vital signs, pulmonary function tests,electrocardiograms, and oxygen saturation and a shift table describingout of range shifts will be provided for clinical laboratory results.Safety ECGs will be classified as normal or abnormal and summarizedusing frequency counts by treatment and time point of collection. Theoccurrence of any change in rhythm from that observed at pre-study orpredose baseline will be noted. Similarly, changes in physicalexamination will be described in the text of the final reports.

Efficacy Outcomes:

For ordinal and categorical outcomes, descriptive statistics will bepresented, with contingency table methods and comparison of frequencyrates calculated. Differences in rates or frequencies by treatment groupwill be calculated, along with 95% confidence intervals (95% CI) fordifferences. Differences in means and CI of mean differences will besimilarly calculated for continuous variables. In the comparison oftreatment related differences for the trajectory of change of renalfunction variables, repeated measures analyses will be employed.

For the dichotomous primary efficacy outcome of DGF, a potential impactof the multiple donor, recipient, and graft variables/characteristicswill be explored by multivariable logistic regression analysis. ITreatment will be included in all models as a dichotomous variable. Inconstructing a final predictor model, individual variables will beincluded if they add to model prediction at significant of p=0.10 orgreater.

Power:

It is estimated from prior renal transplant literature that thefrequency of DGF, as defined above, observed in placebo-treated subjectswill be 30%. It is further hypothesized that inhaled CO Rx will bejudged to have a clinically meaningful impact if it reduces the observedcumulative frequency of observed DGF to 20% in treated individuals, anabsolute decline of 10%, and a relative decline of 33%.

1. A method for improving organ or tissue function or organ or tissue transplant longevity, comprising: administering carbon monoxide (CO)-containing gas a plurality of times to a patient in need of therapy, at a constant alveolar concentration for at least about 30 minutes.
 2. The method of claim 1, wherein the patient is a transplant recipient, optionally selected from kidney, liver, lung, pancreas, heart, bone marrow, and intestine.
 3. The method of claim 1, wherein the patient is experiencing or is at risk of acute rejection, chronic allograft rejection, vascular rejection, or graft versus host disease (GVHD).
 4. The method of claim 1, wherein the patient has a condition selected from a chronic kidney fibrosing condition, a chronic hepatic fibrosing condition, a chronic lung fibrosing conditions, myocardial fibrosis, pancreatic fibrosis, pancreatitis, gastrointestinal fibrosis or strictures, vascular fibrosis or strictures, progressive systemic sclerosis (PSS), scleroderma, esophageal fibrosis or strictures, cirrhosis, atrial fibrosis, Crohn's Disease, Inflammatory Bowel Disease, paralytic ileus, arthritis, arthrofibrosis, and nephrogenic systemic fibrosis.
 5. The method of claim 1, wherein the patient has pulmonary fibrosis, asthma, emphysema, Chronic Obstructive Pulmonary Disease (COPD), pulmonary arterial hypertension (PAH), cystic fibrosis (CF), Acute Respiratory Distress Syndrome (ARDS), bronchiectasis, or Ventilator-Assisted Pneumonia (VA).
 6. The method of claim 1, wherein the patient is experiencing or is at risk of paralytic ileus, necrotizing enterocolitis, Hirschprung's Disease, or toxic megacolon.
 7. (canceled)
 8. The method of claim 2, wherein the kidney donor is an expanded criteria donor.
 9. The method of claim 1, wherein the patient receives CO therapy a plurality of times during a perioperative period.
 10. The method of claim 9, wherein the patient receives inhaled CO therapy substantially before and/or substantially after the perioperative period.
 11. The method of claim 2, wherein the patient receives the inhaled CO therapy intraoperatively.
 12. The method of claim 9, wherein the patient receives from 1 to about 20 administrations of CO therapy prior to surgery.
 13. The method of claim 12, wherein the patient receives CO therapy at from about 0.5 to about 5 hours prior to surgery.
 14. The method of claim 9, wherein the patient receives post-operative CO therapy.
 15. The method of claim 14, wherein the patient receives inhaled CO from 1 to 3 times within the first 24 hours after transplantation.
 16. The method of claim 9, wherein the patient receives inhaled CO from 2 to about 10 times during the perioperative period.
 17. The method of claim 16, wherein the patient receives the first two administrations of inhaled CO separated by at least about 6 hours, the first at the time of transplantation.
 18. The method of claim 9, wherein the patient receives intermittent dosing after the perioperative period for prophylactic treatment.
 19. The method of claim 1, wherein the patient receives inhaled CO therapy at least once per week.
 20. The method of claim 19, wherein the patient receives inhaled CO therapy at least once per week for at least 6 months.
 21. The method of claim 1, wherein the constant alveolar concentration is maintained for at least 45 minutes, or for at least one hour.
 22. The method of claim 1, wherein the CO level in the patient is measured by one or more carboxy-protein levels, level of CO complexed with blood or tissue cytochromes, O₂ or pO₂ optionally in combination with CO-Hb, blood pH, or level of exhaled CO or NO.
 23. The method of claim 22, wherein the CO level in the patient is measured by carboxy-hemoglobin level.
 24. The method of claim 23, wherein the constant alveolar concentration of CO maintains a CO-Hb level of from 8% to 12%.
 25. The method of claim 24, wherein the constant alveolar concentration of CO maintains a CO-Hb level of about 10%.
 26. The method of claim 24, wherein the CO administration comprises at least two concentration levels of CO gas; a high level of CO to quickly reach a target CO level, and a maintenance level of CO to maintain the CO level for a period of time.
 27. The method of claim 26, wherein CO doses are calculated based on relationships defined by the CFK equation.
 28. The method of claim 27, wherein DL_(CO) is calculated based on a CO-Hb measurement taken after from about 15 minutes to about 25 minutes of the first CO dose; and the DL_(CO) calculation is used to predict the dose of CO needed to hit the desired CO-Hb level within a specified period of time.
 29. The method of claim 28, wherein the DL_(CO) calculations are made by a computer system that controls adjustment of the CO dose based on said calculation.
 30. The method of claim 28, comprising: measuring a baseline CO-Hb level in the blood of the patient; identifying a target CO-Hb level; administering to the patient carbon monoxide at a first concentration for an initial time period; measuring the CO-Hb level in the blood of the patient after from about 15 minutes to about 30 minutes of CO exposure; calculating, based on the measured CO-Hb levels and the target CO-Hb level, a dose of carbon monoxide required to attain the target CO-Hb level within a determined time period; and administering to the patient the calculated dose of carbon monoxide for the determined time period to attain the target CO-Hb level in the blood of the patient.
 31. The method of claim 30, further comprising, calculating a dose of CO for maintaining the target CO-Hb level with a constant alveolar concentration of CO, and delivering said constant alveolar concentration.
 32. The method of claim 1, further comprising administering O₂ therapy to the patient to thereby reduce CO toxicity or enhance efficacy of CO therapy.
 33. The method of claim 1, further comprising administering NO therapy to the patient to enhance CO efficacy.
 34. The method of claim 1, wherein the patient is a transplant patient, and immunosuppressive therapy is reduced or eliminated at the initiation of CO therapy.
 35. A system for delivering carbon monoxide therapy, comprising: a source of gas comprising carbon monoxide, a gas metering device operably connected to the CO source, a gas mixing device preparing a constant CO dose with air, a mixed CO-air delivery unit, and a computer system programmed to perform calculations based on the CFK equation, and control adjustment of the CO dose and/or duration of CO dose based on said calculations.
 36. The system of claim 35, wherein the computer system is integrated with the gas metering device.
 37. The system of claim 35, wherein the computer system calculates DL_(CO) based on a baseline CO-Hb level, a measured CO-Hb level at a timepoint during CO administration, and delivered or inspired CO.
 38. The system of claim 37, wherein the computer system controls the CO concentration and/or duration of CO dose based on the calculated DL_(CO) and target CO-Hb level.
 39. The system of claim 35, wherein CO-Hb values are input by a user or input automatically through communication with sensor or diagnostic instruments.
 40. The system of claim 35, wherein CO gas for inspiration is premixed and delivered as a constant concentration, independent of respiratory rate, FRC, or tidal volume. 